1. Field of the Invention
The present invention relates to isolated polypeptides having aminopeptidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Various food products, e.g., soups, sauces and seasonings, contain flavoring agents obtained by hydrolysis of proteinaceous materials. This hydrolysis is conventionally accomplished using strong hydrochloric acid, followed by neutralization with sodium hydroxide. However, such chemical hydrolysis leads to severe degradation of the amino acids obtained during the hydrolysis, and also to hazardous byproducts formed in the course of this chemical reaction. Increasing concern over the use of flavoring agents obtained by chemical hydrolysis has led to the development of enzymatic hydrolysis processes.
Enzymatic hydrolysis of proteinaceous materials aims at obtaining a high degree of hydrolysis (DH), and this is usually attained using a complex of unspecific acting proteolytic enzymes (i.e., unspecific-acting endo- and exo-peptidases). For example, WO 94/25580 describes a method for hydrolyzing proteins by use of an unspecific acting enzyme preparation obtained from Aspergillus oryzae. Specific acting proteolytic enzymes have not been used for this purpose because such enzymes only lead to an inadequate degree of hydrolysis.
Polypeptides having aminopeptidase activity catalyze the removal of one or more amino acid residues from the N-terminus of peptides, polypeptides, and proteins. Such polypeptides are classified under the Enzyme Classification Number E.C. 3.4.11.xe2x80x94of the International Union of Biochemistry and Molecular Biology.
WO 96/28542 discloses an aminopeptidase which has a moleculer weight of 35 kDa. JP-7-5034631 (Noda) discloses a leucine aminopeptidase obtained from yellow koji mold, which includes Aspergillus oryzae JP-7-4021798 (Zaidan Hojin Noda Sangyo) discloses the production of miso by adding a leucine aminopeptidase II prepared by cultivating a number of strains, including Aspergillus oryzae strain 460 (ATCC 20386) and strain IAM 2616. Aspergillus oryzae strain 460 is known to produce a number of leucine aminopeptidases of which three have a molecular weight of 26.5, 56, and 61 kDa by gel filtration (Nakada et al., 1972, Agricultural and Biological Chemistry 37: 757-765; Nakada et al., 1972, Agricutural and Biological Chemistry 37: 767-774; and Nakada et al., 1972, Agricultural and Biological Chemistry 37: 775-782; respectively). Penicillium citrium produces an intracellular leucine aminopeptidase with a molecular weight of 65 kDa by SDS-PAGE (Kwon et al., 1996, Journal of Industrial Microbiology 17: 30-35). Lactobucillus helveticus produces an endopeptidase of about 70 kDa which hydrolyzes peptides with 3-34 amino acid residues but not protein (EP 733,703). Pseudomonas fluorescens produces an intracellular aminopeptidase of about 50 kDa (Gobbetti et al., 1995, Journal of Dairy Science 78: 44-54). Listeria monocytogenes is reported to produce a glycine aminopeptidase (U.S. Pat. No. 5,330,889). WO 96/06175 discloses enzymes capable of degrading and detoxifying fumonisins. Telesnina et al., (1992, Antibiotiki I Khimioterapiya 37: 14-16) disclose the isolation of Xanthomonas rebrilineans protoplasts and their use in the study of aminopeptidase localization.
WO 97/04108 (Roehm) discloses DNA encoding an Aspergillus sojae leucine aminopeptidase. Chang and Smith (1989, Journal of Biological Chemistry 264: 6979-6983) disclose the molecular cloning and sequencing of a gene encoding a vacuolar aminopeptidase from Saccharomyces cerevisiae. Chang et al. (1992, Journal of Biological Chemistry 267: 8007-8011) disclose the molecular cloning and sequencing of a gene encoding a methionine aminopeptidase from Saccharomycse cerevisiae. 
The production of protein hydrolysates with desirable organoleptic properties and high degrees of hydrolysis generally requires the use of a mixture of peptidase activities. It would be desirable to provide a single component peptidase enzyme which has activity useful for improving the organoleptic properties and degree of hydrolysis of protein hydrolysates used in food products either alone or in combination with other enzymes.
It is an object of the present invention to provide improved polypeptides having aminopeptidase activity and nucleic acid encoding the polypeptides.
The present invention relates to isolated polypeptides having aminopeptidase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 50% identity with amino acids 33 to 674 of SEQ ID NO:2;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or (ii) its complementary strand; or a subsequence thereof of at least 100 nucleotides;
(c) an allelic variant of (a) or (b);
(d) a fragment of (a), (b), or (c), wherein the fragment has aminopeptidase activity; and
(e) a polypeptide which (i) has aminopeptidase activity in the pH range between pH 5.0-8.5 measured at 37xc2x0 C., (ii) has an isoelectric point in the range of 7.4-8.5; (iii) has aminopeptidase activity in the temperature range of 20-55xc2x0 C., measured at pH 7.5 using Gly-pNA in Tris-HCl buffer; (iv) hydrolyzes Ala-pNA, Gly-pNA, Leu-pNA, Glu-pNA, Asp-pNA, Lys-pNA, Ile-pNA and Val-pNA; (v) does not hydrolyze Phe-pNA nor Pro-pNA; (vi) is not inhibited by phenylmethanesulfonyl fluoride, slightly inhibited by EDTA, di-isopropyl fluoro phosphate, p-chloromercuribenzoic acid and iodoacetic acid, completely inhibited by o-phenanthroline, and/or (vii) is obtained from a strain belonging to Sphingomonas and has a molecular mass of 67xc2x15 kDa.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.